RU2795090C1 - Method for predicting the recurrence of early preeclampsia by markers of endothelial dysfunction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to obstetrics, and can be used to predict the recurrence of early preeclampsia by markers of endothelial dysfunction. The level of endothelin-1 and the level of microvesicles (EVs) of endothelial origin 0.1–1 μm in size are determined in blood plasma at gestation periods of 11–13 weeks using antibodies to CD144. The risk of developing early preeclampsia is predicted at an endothelin-1 level of more than 0.497 pmol/ml and an EVs level of endothelial origin of more than 0.97 CD144+ events/µl.

EFFECT: method provides the possibility of predicting the recurrence of early preeclampsia in patients at risk, allowing timely stratification of pregnant women into a group for additional examination methods and preventive measures, by determining the concentration of endothelin-1 in peripheral blood plasma and measuring the number of CD144 positive (+) events (EVs of endothelial origin) in the volume of platelet-free blood plasma during 11–13 weeks of gestation.

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Description

The invention relates to medicine, in particular to obstetrics, and can be used to predict the recurrence of early preeclampsia according to biomarkers of systemic endotheliosis, namely the level of endothelin-1 and microvesicles of endothelial origin at 11-13 weeks of gestation.

Preeclampsia (PE) is a specific complication of pregnancy associated with high blood pressure, increased renal vascular resistance, and endothelial dysfunction. It has been determined that PE is one of the main causes of maternal and perinatal morbidity and mortality, and pathogenetically substantiated therapeutic interventions have not yet been developed due to an incomplete understanding of the mechanisms involved in the pathophysiology of PE (Sava RI, March KL, Pepine CJ. Hypertension in pregnancy : Taking cues from pathophysiology for clinical practice Clin Cardiol 2018 Feb;41(2):220-227 doi: 10.1002/clc.22892.).

At the same time, recent studies show that the primary link in the pathogenesis of PE is maternal endothelial dysfunction (Sidorova I.S., Nikitina N.A. Preeclampsia as gestational immune complex complement-mediated endotheliosis. Russian Bulletin of an obstetrician-gynecologist. 2019; 19(1): 5 -11. https://doi.org/10.17116/rosakush2019190115.), characterized by dysfunction of several signaling pathways, including the endothelin-1 system and abnormal expression of endothelial-derived extracellular microvesicles (EVs). A meta-analysis of 16 published cohort studies, including 1739 patients with realized hypertension and 409 controls, showed that plasma concentrations of endothelin-1 are increased in pregnant women with hypertension compared with normal blood pressure controls (Lu YP, Hasan AA, Zeng S, Hocher B. Plasma ET-1 Concentrations Are Elevated in Pregnant Women with Hypertension -Meta-Analysis of Clinical Studies Kidney Blood Press Res. 2017;42(4):654-663 doi: 10.1159/000482004.). The most likely cause of increased endothelin-1 biosynthesis is placental ischemia due to inadequate invasion of syncytiotrophoblast villi (Bakrania BA, Spradley FT, Satchell SC, Stec DE, Rimoldi JM, Gadepalli RSV, Granger JP. Heme oxygenase-1 is a potent inhibitor of placental ischemia-mediated endothelin-1 production in cultured human glomerular endothelial cells Am J Physiol Regul Integr Comp Physiol 2018 Mar 1;314(3):R427-R432 doi: 10.1152/ajpregu.00370.2017).

EVs of endothelial origin are considered as key biological markers of endothelial dysfunction/destruction, acting as messengers of intercellular communication in the pathogenesis of systemic endotheliosis, a clinical example of which is early PE (Brewster LM, Garcia VP, Levy MV, Stockelman KA, Goulding A, DeSouza NM, Greiner JJ, Hijmans JG, DeSouza CA Endothelin-1-induced endothelial microvesicles impair endothelial cell function J Appl Physiol (1985) 2020 Jun 1;128(6):1497-1505 doi: 10.1152/japplphysiol.00816.2019).

Despite the fact that patients with early PE in their personal anamnesis belong to the high-risk group for recurrence, in which a set of preventive measures and in-depth follow-up are carried out, the recurrence of the disease reaches 10-25%, which suggests the search for new biomarkers and targets for therapeutic opportunities (Brouwers L , van der Meiden-van Roest AJ, Savelkoul C, Vogelvang TE, Lely AT, Franx A, van Rijn BB Recurrence of pre-eclampsia and the risk of future hypertension and cardiovascular disease: a systematic review and meta-analysis BJOG. 2018 Dec;125(13):1642-1654 doi: 10.1111/1471-0528.15394).

There is a known method for predicting the risk of developing PE in women of Russian nationality who are natives of the Central Black Earth region of Russia (RF patent, 2016: RU 2642939) by analyzing the polymorphism of metalloproteinase-1 (MMP-1) (rs 1799750) and assessing the risk of developing PE according to equations of a linear discriminant function, including such variables as body mass index, history of PE, presence of gynecological pathology, presence of sexually transmitted infections, systolic blood pressure (BP), diastolic BP, genetic variant for the MMP-1 locus.

The development of PE is predicted by the sum of the scores obtained in the formula of binary logistic regression equations. The disadvantage of this method is the need for complex mathematical calculations, drawing up equations of a linear discriminant function. The informativeness of the proposed method has been proven only for patients who are natives of the Central Black Earth region of Russia.

There is a known method for predicting early PE at 11-13 weeks of gestation (RF Patent, 2018: RU 2732489) by determining P1GF (placental growth factor) and sVEGF R1 (soluble vasculoendothelial growth factor receptor-1) in the peripheral blood of women at gestational age 11 -13 weeks. When registering P1GF more than 60 pg/ml in combination with sVEGF R1 more than 2000 pg/ml, an increase in the risk of developing early preeclampsia is predicted. The disadvantage of this method is the high cost, as well as the specificity of the above markers in relation to fetal growth retardation (FGR), as well as the formation of placental insufficiency, the presence of which may complicate the diagnosis of PE.

There is a known method for predicting severe PE (RF patent, 2020: RU 2739694), based on the calculation of the prognostic index for the development of PE according to the formula: RIPE= -2.42X1-3.17X2+1.48X3+0.1X4-0.1X5-7 ,35X6+1.93X7-32.3, where P1PE is a prognostic index for the development of severe preeclampsia; X1-presence of anemia (1 - the patient has anemia (hemoglobin in the 1st trimester <110 g/l), 0 - no anemia); X2 - heredity burdened by cardiovascular pathology (1 - burdened heredity, 0 - unburdened heredity); X3 - APTT level in the 1st trimester, s; X4 - ACT level in the 1st trimester, U/l; X5 - creatinine level in the 1st trimester, µmol/l; X6- MHO in the 1st trimester, arb. units; X7 - level of PAPP-A at 11-13.6 weeks, MoM; Constant = - 32.3. When RIPE > 0, a low risk of developing severe preeclampsia is predicted, and when RIPE ≤ 0, a conclusion is made about a high risk of developing severe PE. The disadvantage of the claimed method is the need to perform a significant number of laboratory tests, the lack of references to the list of cardiovascular diseases in a pregnant woman, which can reduce the information content of the method, and the need for complex mathematical calculations.

The prototype of the proposed method is the patent of the Russian Federation RU 2693412 (2018) "Method for predicting early and late preeclampsia", in which the authors, by quantitative determination of the concentration of tumor necrosis factor alpha (TNFα), placental growth factor (GFR) and placental alpha-1 microglobulin in blood plasma (PAMG), insulin resistance index (HOMA-IR), insulin and leptin levels at 11-14 and 18-21 weeks of gestation predict early and late PE, respectively. With the value of these indicators in terms of 11-14 weeks and 18-21 weeks of pregnancy, respectively: the concentration of TNFα (pcg/ml) - 44±5.1 and 91±8.7; FRP concentrations (pg/ml) - 84±11 and 263±22; PAMG concentrations (ng/ml) - 8.6±2.2 and 72.4±7.6; HOMA-IR value - 1.52±0.13 and 3.64±0.36; insulin levels (pmol/ml) - 59.7±5.6 and 152.3±7.8; leptin levels (ng/ml) -34.1±3.3 and 48.3±4.4 predict early onset PE; with the value of these indicators at the indicated terms of pregnancy, respectively: the concentration of TNFa (pcg/ml) - 16±2.8 and 86±7.5; FRP concentrations (pg/ml) -114±12 and 272±26; PAMG concentrations (ng/ml) - 7.7±2.1 and 75.6±7.9; HOMA-IR value - 0.93±0.08 and 3.58±0.33; insulin levels (pmol/ml) - 33.7±3.4 and 144.6±7.4; leptin levels (ng/ml) - 20.6±2.4 and 44.5±4.1 predict late onset PE.

The disadvantage of the method is the need to conduct a study twice during pregnancy at 11-14 and 18-21 weeks. In addition, the reference values for the physiological course of pregnancy, which are the basis of the invention, are specific to the recruited sample and may differ from those in the population.

The authors propose a simple, accessible method for predicting the recurrence of early PE in patients at risk, allowing timely stratification of pregnant women into a group for additional examination methods and preventive measures.

The technical result of the proposed method is achieved by determining the concentration of ET-1 in peripheral blood plasma and measuring the number of CD144 positive (+) events (EVs of endothelial origin) in the volume of platelet-free blood plasma during gestation of 11-13 weeks.

The method is carried out as follows.

To determine the concentration of ET-1, blood for research is taken in the morning, on an empty stomach, from the cubital vein into 3.0 ml VACUETTE tubes containing EDTA. Blood must be processed within 1 hour of collection. Centrifugation is carried out at 1500g for 15 minutes at room temperature to obtain platelet-poor plasma (PPP). If necessary, platelet-poor plasma samples are transferred to eppendorfs and frozen, followed by storage at -50°C until analysis (but not more than 6 months). On the day of analysis, plasma samples are thawed (if they have been frozen) at +37°C in a water bath.

This test is based on a quantitative sandwich enzyme-linked immunosorbent assay. The microplate wells are coated with specific monoclonal antibodies to endothelin-1. During the reaction, standards and samples are added to the wells of the microplate, and the endothelium-1 present in the sample binds to the immobilized antibodies. After washing, unbound components are removed, and enzyme-conjugated monoclonal antibodies to endothelin-1 are added to the wells.

After a second wash and removal of the unbound enzyme-antibody conjugate, a substrate solution is added which reacts with the enzyme to form a color complex. The color intensity of the solution is directly proportional to the concentration of endothelin-1 present in the sample. The color reaction is stopped with a stop solution and the color intensity is measured on a microplate photometer.

To measure the number of CD144 positive (+) events (EVs of endothelial origin), blood sampling for research is carried out in the morning, on an empty stomach, from the cubital vein into 4.5 ml VACUETTE tubes containing buffered sodium citrate solution 3.2%, the ratio of blood volumes and sodium citrate - 9:1. Blood must be processed within 1 hour of collection. Further, by successive centrifugations, platelet-free plasma (PFP or PTP) is obtained, which is subjected to further analysis. First, centrifugation is performed at 2500×g for 15 minutes at room temperature to obtain platelet-poor plasma (PPP). The top layer (3/4) of PRP is centrifuged twice more at 2500 x g for 15 minutes at room temperature, removing 3/4 of the top layer of supernatant at each step to obtain platelet-free plasma (PFP or PFP), which can be immediately analyzed.

If necessary, platelet-free plasma samples can be frozen at the boiling point of liquid nitrogen (-196°C) and then stored at -70°C until analysis (but not more than 6 months). On the day of analysis, frozen plasma samples are thawed at +37°C in a water bath, transferred from cryotubes to microcentrifuge tubes, and additional centrifugation 3000xg for 10 minutes is carried out. After centrifugation, the supernatant (3/4 of the upper layer) is used for analysis.

Preparation of conjugated antibodies and instrument setup.

For staining of endothelial extracellular vesicles according to this invention, it is proposed to use antibodies against CD144 (VE-cadherin, or cadherin 5 type 2). Anti-CD144 antibodies can be conjugated to any commercially available fluorophore that provides a good signal for low abundance events. Before working with antibodies, their preparation is necessary to eliminate noise: antibodies are diluted 20 times with phosphate-buffered saline and subjected to centrifugation 20,000 × g - 12 minutes to remove possible conjugates.

Staining is carried out by mixing 5 µl of conjugated antibodies with 50 µl of the sample, mixing and subsequent exposure in a dark place for 20-30 minutes. As a negative control, it is acceptable to use both an unstained sample in the same dilution as the stained sample, and the corresponding isotype control. Direct measurement of events can be carried out on any flow cytometer with an event detection limit of at least 200 nm. For example, CytoFlex (Beckman Coulter, USA), which uses side scatter at a shorter wavelength of 405 nm (violet laser) as a trigger signal, which increases the sensitivity and resolution of the objects under study, making it possible to detect particles with a diameter of less than 500 nm (detection limit at level 150-200 nm). To attribute CD144+ events to extracellular vesicles, the size parameter (0.1-1 µm) is used, the gate for which is formed during the use of fixed-size microbeads with an attached fluorescent label (0.1-1.0 µm). For example, Flow Cytometry Sub-micron Particle Size Reference Kit, Thermoflsher, USA, with a set of calibration beads 0.1, 0.2, 0.5, and 1.0 µm, which set the approximate gate in the range from 0.1 to 1 µm.

Mandatory in the described method is to check for a possible contribution to the "noise" for phosphate-buffered saline, as well as for antibodies diluted in phosphate-buffered saline in the same proportions as the stained samples. Only after that you can proceed to the measurement for unstained samples, on which the border for the “CD144-” gate is set. Accordingly, all events that exceed the negative gate in signal value are referred to as positive events.

Stained samples after 20-30 minutes of incubation are shaken again and the measurement is carried out after diluting the sample with phosphate-buffered saline sufficient to avoid the swarm effect (“swarm effect”) or coincidence (coincidence). In each analysis, 1 million of all events are recorded at a speed of 10 μl/min, after making sure that there are no objects larger than 1000 nm in the analyzed area. After measuring the stained sample, a test with Triton XI00 (detergent that destroys the membranes of eukaryotic cells) can optionally be carried out.

For statistical processing, the parameter of the number of events (extracellular events) per 1 μl was used.

To confirm the effectiveness of the proposed method, a prospective observational study was conducted, with the inclusion of 127 patients with a history of early severe preeclampsia, who, according to the outcomes of the observed pregnancy, were divided into 2 groups: with a favorable outcome of this pregnancy (comparison group n = 59), and with a relapse of severe PE in current pregnancy (main group n=38). 30 patients were excluded from the observation group by the time the comparison groups were formed for objective reasons (17 women had a non-developing pregnancy, 9 women took low molecular weight heparins from the second trimester of pregnancy, 3 patients had a premature detachment of a normally located placenta, 1 woman refused to participate in the study ).

Criteria for inclusion in the study: singleton spontaneous pregnancy, age of the patient from 18 to 35 years, history of early severe PE.

Criteria for exclusion from the study: pregnancy in assisted reproductive technology programs, multiple pregnancy, family and personal aggravated thrombotic and hemorrhagic history, somatic pathology (hypertension, diabetes mellitus, obesity, systemic lupus erythematosus, antiphospholipid syndrome). It should be noted that all pregnant women from 12 weeks of pregnancy received acetylsalicylic acid in a daily dose of 150 mg, according to clinical recommendations.

The comparison groups formed at the end of gestation were comparable in terms of clinical characteristics and social status. The median age of patients in the control group was 31 [95% CI 29.0-34] years, the main - 28.4 [95% CI 25.6-33.4] years.

The median (Me) of the level of ET-1 during examination at 11-13 weeks of gestation in the control group was 0.42 pmol/ml (95% CI: 0.29-0.61), in the main 0.92 pmol/ml (95% CI: 0.81-1.11), the difference is statistically significant (p=0.0003) (Fig. 1). To determine the threshold value of ET-1 in blood plasma and the degree of prognostic information of the test, an ROC analysis was performed. It was found that when the level of ET-1 in blood plasma is >0.497 pmol/ml, the number of correctly classified positive cases is 100%, correctly classified negative cases is 62.5% (Fig. 2).

The median (Me) level of EVs of endothelial origin during examination at 11-13 weeks of gestation in the control group was 0.40 events/µl (95% CI: 0.17-0.44), in the main 1.77 events/µl ( 95% CI: 1.67-2.97), the difference is statistically significant (p<0.0001) (Fig. 3). To determine the threshold value of the level of EVs of endothelial origin in blood plasma and the degree of prognostic information of the test, a ROC analysis was performed. It was determined that when the value of the level of EVs in blood plasma is >0.97 events/µl, the number of correctly classified positive cases is 100%, correctly classified negative cases is 100% (Fig. 4).

Clinical observation 1

I.S.N. - multiparous, multiparous 30 years. Menstrual function is not disturbed. In 2019, premature birth at 28 weeks' gestation by surgery due to severe preeclampsia (BP 210/120, protein in a single portion of urine 6.0 grams/liter). A live male fetus weighing 720 grams was born, died on the 3rd day of life. This (analyzed) pregnancy is the second, spontaneous, desired. Has come on 2 months of regular sexual life without contraception after the stage of preconception preparation. When taken to the dispensary, the patient's height was 155, weight 44 kg, BMI=18.3. I attended counseling sessions regularly. During the examination in the first trimester, no deviations in laboratory parameters were registered.

First ultrasound screening at 11.5 weeks gestation. No ultrasound markers of chromosomal pathology were found. Ultrasound anatomy of the fetus without pathology. The risk of developing obstetric complications in the Astraia program was calculated as high (the risk of PE up to 37 weeks of gestation is 1 out of 67, the risk of fetal growth retardation up to 37 weeks of gestation is 1 out of 54). The level of ET-1 at 12 weeks of pregnancy was 0.98 pmol/ml, the level of EVs of endothelial origin was 1.94 sob/µl. During the second ultrasound screening at a gestational age of 19.5 weeks: ultrasound markers of chromosomal pathology were not detected. Ultrasound anatomy of the fetus without pathology. Fetometry indicators are within the reference values. According to Doppler, pathological blood flow in the uterine arteries, vessels of the umbilical cord was not registered.

At 29 weeks, against the background of complete well-being, a severe headache appeared, which was not stopped by taking analgesics (I took it on the advice of relatives). When applying to the hospital AD 210/120; proteinuria in a single portion of urine 7 grams / liter. She was transferred to the intensive care unit (diagnosis: pregnancy 29.1 weeks; severe preeclampsia), with stabilization of the condition (after 4 hours), she was urgently delivered by the abdominal route. A premature boy was born, weighing 1450. Blood loss during childbirth was 650 ml. Stabilization of the patient on the 3rd day after delivery. Transferred with the child to the second stage of nursing newborns. clinical observation 2.

Z.T.G. - multiparous, multiparous, 35 years old. Menstrual function is not disturbed, gynecological diseases are absent. She has a history of preterm labor at 30 weeks' gestation due to severe preeclampsia. A girl was born, weighing 1380 grams. Denies somatic diseases. Pregnancy desired, planned, came immediately after the abolition of contraception. When taken to the dispensary, the patient's height was 161, weight 64 kg, BMI=24.69. I attended counseling sessions regularly. During the examination in the first trimester, no deviations in laboratory parameters were registered.

The first ultrasound screening at 11.5 weeks' gestation: CTE 50.2 mm, corresponds to 11.5 weeks' gestation. No ultrasound markers of chromosomal pathology were found. Ultrasound anatomy of the fetus without pathology. Doppler characteristics of uteroplacental blood flow are normal. The risk of developing obstetric complications in the Astraia program was calculated as high (the risk of PE up to 37 weeks of gestation is 1 out of 82, the risk of fetal growth retardation up to 37 weeks of gestation is 1 out of 68). The level of ET-1 at 12 weeks of pregnancy was 0.47 pmol/ml, the level of EVs of endothelial origin was 0.38 sob/µl.

During the second ultrasound screening at a gestational age of 20.1 weeks: fetometrically, the fetus corresponds to a period of 20.1 weeks. No ultrasound markers of chromosomal pathology were found. Ultrasound anatomy of the fetus without pathology. The course of pregnancy is favorable.

At 38 weeks, a planned operative delivery was performed due to the presence of a scar on the uterus after a cesarean section in history and the woman's refusal to give birth through the natural birth canal. A girl was born, weighing 3100 grams 50 cm. On the 5th day they were discharged home with a child from the maternity hospital.

Claims (1)

A method for predicting the recurrence of early preeclampsia by markers of endothelial dysfunction, which includes determining the level of endothelin-1 and the level of microvesicles (EVs) of endothelial origin with a size of 0.1-1 μm in blood plasma at 11-13 weeks of gestation using antibodies to CD144, characterized by the fact that that the risk of developing early preeclampsia is predicted at an endothelin-1 level of more than 0.497 pmol/ml and an endothelial-derived EVs level of more than 0.97 CD144+ events/µl.

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